Nonalcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease worldwide. It is now estimated to affect close to 30% of the adult US population. NAFLD represents a wide spectrum of conditions ranging from simple fatty liver which in general follows a benign non progressive clinical course, to steatohepatitis or NASH, a more serious form of NAFLD characterized by hepatocellular apoptosis, liver injury and inflammation that may progress to cirrhosis and end-stage liver disease. At present time, a liver biopsy remains the only reliable way to diagnose NASH and establish the severity of disease. Current non-invasive clinically available tests lack accuracy and reliability. In light of the dramatic increase in the prevalence of NAFLD in conjunction with the significant research effort in developing novel therapies targeted to those patients with NASH, non invasive, simple, reproducible and reliable mechanism-based biomarkers which can not only help in the diagnosis of NASH, but also be useful endpoints for assessment of treatment response and prognosis are urgently needed. Thus, the overall objectives of this proposal are to define the molecular mechanisms contributing to liver cell death and the link to liver damage and disease progression in NAFLD, as well as to establish a mechanism-based biomarker for patients with this condition. Based on extensive preliminary data, we propose the novel central hypothesis that in NAFLD, hepatocyte caspase-3 activation plays a mechanistic role in the pathogenesis of liver damage and plasma caspase-cleaved Cytokeratin (CK)-18 fragment levels is an ideal biomarker for patients with this condition. The established collaboration and support from the Nonalcoholic Steatohepatitis NIH Clinical Research Network (NASH CRN), the Bariatric and Metabolic Institute at the Cleveland Clinic, and the Case Alcoholic Steatohepatitis (CASH) Registry as well as the availability of caspase 3 genetically deficient mice will serve as critical tools for these studies. This proposal is in response to Program Announcement PA-07-052 Development of Disease Biomarkers. Our proposal has the following Specific Aims; First, we will determine the utility for non-invasive quantification of caspase-generated CK-18 fragment levels in blood for NASH diagnosis, monitoring disease progression and response to therapeutic interventions over time. Second, we will establish the temporal and causal relationship of caspase activation and CK-18 cleavage in hepatocytes, with increase in plasma CK-18 fragment levels, and disease progression by using human samples as well as in vivo dietary models of NAFLD and in vitro cell models of lipid overloading. The proposal is innovative technically and conceptually as it tests new concepts for lipid induced hepatotoxicity using sophisticated technologies. Moreover, The results of this proposal may not only bring new insights to the specific mechanisms responsible for disease progression in NAFLD, but also could translate into the first reliable noninvasive biomarker clinically available with a tremendous positive impact in several aspects of the care of patients with this highly common and potentially serious condition. PUBLIC HEALTH RELEVANCE: Nonalcoholic fatty liver disease (NAFLD) is a serious public health problem. It is now estimated to affect 30% of adults and 10% of children in the U.S. NAFLD represents a wide spectrum of conditions ranging from fatty liver which in general follows a benign non progressive clinical course, to steatohepatitis or NASH, a more serious form of NAFLD that may progress to cirrhosis and end-stage liver disease. At present time, an invasive liver biopsy remains the only reliable way to diagnose NASH and establish the severity of disease. Our preliminary studies suggest that liver biopsy may be avoidable with the development of a new biomarker that can be used to differentiate NASH from fatty liver in patients with suspected NAFLD, using a simple blood sample. The biomarker is based on caspase 3-generated Cytokeratin 18 (CK-18) fragments, which are elevated in NASH compared with fatty liver. The results of this proposal may not only bring new insights to the specific mechanisms responsible for disease progression in NAFLD, and thus suggesting novel therapeutic strategies, but also may result in a relatively short term, in the development and validation of the first clinically available, reliable, non- invasive NAFLD biomarker. The potential impact that such a biomarker could have in the care of patients for this highly common and potentially serious condition is great.